1. Field of the Invention
The invention relates to the chemistry of biologically active compounds, namely, to a new method to prepare water-soluble mono-PEGylated tetrapyrrole derivatives, particularly chlorin, bacteriochlorin, pheophorbide and bacteriopheophorbide derivatives of types 1, 2 and 3. The compounds of the present invention can be used as photosensitizers for the photodynamic therapy of cancer, infections and other diseases as well as for light irradiation treatments in other cases. Wherein B is a ring having the structure: Wherein:                R1=—CH=CH2, —CH(OAlk)CH3, —CHO, —C(O)CH3, —CH2CH3, —CH(Alk)CH(COAlk)2, —CH2CH(COAlk)2, —CH(Alk)CH2COAlk, —CH(Alk)CH2CH(OH)CH3, and —CH2CH2CH(OH)CH3;        R2=—CH3, —CHO, —CH(OH)Alk, —CH═CHAlk, CH2OH, and CH2OAlk;        R3=—OH, —OAlk, —NH-Alk, —NR8—R9—R10, —NH(CH2)m—R11—R9—R10;        R4=—OH, —OAlk, —NH-Alk, —NR8—R9—R10, —NH(CH2)m—R11—R9—R10;        R5=OH, —OAlk, —NH-Alk, —NR8—R9—R10, —NH(CH2)m—R11—R9—R10;        R6=H and —COOAlk;        R7=NR8—R9—R10, —NH(CH2)m—R11—R9—R10;        R8=H and -Alk;        R9=—(CH2CH2O)nCH2CH2—;        R10=—OH, —OAlk, —NH2, —NHAlk, —NHAcyl, —NAcyl2, —NR12R13, —COR14, —OCH2COR14;        R11=—CH2CONR8—, —NHCOO—;        R12=H and -Alk;        R13=H and -Alk; and        R14=—OH, —OAlk, —NR12R13;Wherein:        m=2-12;        n=8-500; and        Alk=an alkyl substituent.        
2. Information Disclosure Statement
Photodynamic therapy (PDT) is one of the most promising new techniques now being explored for use in a variety of medical applications, and particularly is a well-recognized treatment for the destruction of tumors (E. D. Sternberg et al, “Porphyrin based photosensitizers for Use in Photodynamic Therapy,” Tetrahedron 54 (1998) 4151-4202).
Criteria are provided which a compound has to meet to at least some extent in order to be successfully used in PDT. (R. Bonnett, “Photodynamic Therapy in Historical Perspective”, Rev. Contemp. Pharmacother. 1999, 10, 1-17) They are the following:                1. high quantum yield of reactive species, such as singlet —oxygen or radicals;        2. relatively low toxicity to the subject;        3. capability of being activated by radiation with a high wavelength (preferably in the red or near infra-red region of the spectrum), which is able to penetrate more deeply into the tissues as compared to radiation with a shorter wavelength;        4. selective accumulation by cells that are responsible for a given pathological condition and rapid elimination from the tissues that are not affected by the pathological condition;        5. potential for being conjugated to macromolecular carries, albeit maintaining the characteristics of photosensitization efficiency, and        6. solubility in suitable solvents to facilitate administration to a patient and physiological uptake and transport within the patient's body.        
Tetrapyrroles are compounds widely used in PDT. A major problem in the pharmaceutical application of tetrapyrroles is their low solubility in physiological solutions. This renders it difficult to prepare effective pharmaceutical grade injectable solutions for PDT and other applications.
Methods to prepare water soluble tetrapyrrole derivatives for PDT are known in the art. U.S. Pat. No. 5,330,741 by Smith et al discloses a method to prepare trisodium lysyl-chlorin p6 involving the reaction between purpurin 18 methyl ester, resulting from methyl pheophorbide a transformation, and aqueous lysine in methylene chloride in the presence of pyridine. The mixture is stirred at room temperature for 12 hours, followed by the removal of the solvents in a high vacuum. The so prepared crude product is purified by reversed phase high performance liquid chromatography (HPLC) and subsequently lyophilized. To prepare an injectable solution for the PDT of cancer, the preparation is first dissolved in phosphate buffer solution and then 0.1 N sodium hydroxide is added. The pH value of the solution is adjusted to pH 7.35 using 0.1 N HCl followed by sterility filtration through a microporous filter. Drawbacks of the above-mentioned method include a lack of reproducibility and difficulty in the work-up and utilization of toxic reagents, which make it hardly appropriate for pharmaceutical manufacturing. Additionally, the prepared water soluble product of interest is stable in an aqueous solution for only 24 hours at 4° C. in the dark, and in solid form for up to 4 months at 4° C. in the dark [M. W. Leach, R. J. Higgins, J. E. Boggan, S.-J. Lee, S. Autry, K. M. Smith, Effectiveness of a Lysylchlorin p6/Chlorin p6 mixture in Photodynamic Therapy of the Subcutaneous 9L Glioma in the Rat. Cancer Res., 1992, 52, 1235-1239; U.S. Pat. No. 5,330,741].
There is a method to prepare a water-soluble sodium salt of pheophorbide a (4), described in U.S. Pat. No. 5,378,835 by Nakazato. According to this invention, pheophorbide a (5) is dissolved in diethyl ether, and a very diluted solution of alkali in n-propanol, iso-propanol or in their mixture is added dropwise and very slowly to the solution. The reaction is maintained up to the complete precipitation of pheophorbide a salt (4), which is separated by centrifugation and dried in vacuo. Then the product is dissolved in water resulting in a solution with concentration 0.5% and pH 9.2-9.5 that is then diluted with a phosphate buffer with pH 7.4-7.8. The drawback of the method described by Nakazato is the fact that a concentrated (>1%) injectable pheophorbide a solution in water cannot be generated by this technique. Additionally, the authors of the present invention observed the chemical instability of such salts when stored dry, and their inability to completely dissolve in water after having been stored in the dry state. 
There is a method disclosed in Russian Patent No. RU2144538 by G. V. Ponomarev et al to prepare water-soluble complexes of chlorin e6 (6) with spacious organic amines including N-methyl-D-glucosamine by a multi-step straightforward sequence of chemical reactions including preparation of chlorophyll a from Spirulina Platensis cyanobacteria biomass, further conversion into chlorin e6 according to standard procedures [S. Lötjönen, P. H. Hynninen, An improved method for the preparation of (10R)-and (10S)-pheophytins a and b. Synthesis. 1983, 705-708; P. H. Hynninen, S. Lötjönen, Preparation of phorbin derivatives from chlorophyll mixture utilizing the principle of selective hydrolysis. Synthesis. 1980, 539-541; S. Lötjönen, P. H. Hynninen, A convenient method for the preparation of wet chlorin e6 and rhodin g7 trimethyl esters. Synthesis, 1980, 541-543] with an overall yield exceeding 50% after precipitation of chlorin e6 (6) by way of stepwise addition of water to its acetone solution, followed by separation by centrifugation and 3-fold washing with water and subsequent treatment of wet chlorin e6 with water solution of 2 g-eq. spacious organic amine. Unfortunately the samples of water soluble salts of chlorin e6 prepared according to the above method contain a variety of impurities of non-tetrapyrrole and tetrapyrrole types which can't be separated from the target chlorin e6 product using conventional procedures.
There is a method to prepare highly pure pharmaceutical-grade water-soluble tetrapyrrole derivatives comprising the steps of: one or two step direct acidic alcoholysis of biological raw material giving crystalline alkyl pheophorbide, conversion of the obtained alkyl pheophorbide into pheophorbide, and reacting of the latter with a hydrophilic organic amine in a medium selected from a group consisting of water and an aqueous organic solution (U.S. patent application Ser. No. 10/151,764 by Nifantiev et al). The hydrophilic organic amine is selected from the group consisting of N-methyl-D-glucamine, aminoalkyl glycosides, tris(hydroxymethyl)aminomethane (“TRIS”) and derivatives thereof, aminoacids and oligopeptides.
Obtaining water-soluble compounds for pharmaceutical applications by means of so-called PEGylation, that is by direct or indirect (via linker) attachment of polyethylene glycol chains (PEG), is known in the art. PEG is non-toxic, increases the water solubility of therapeutic molecules, and alters the biodistribution, which can result in a favorable pharmacokinetic profile (International Application No. WO 01/66550 by Bradley et al).
Water-soluble PEGylated compounds for PDT are disclosed in U.S. Pat. No. 5,622,685 by Sinn et al, wherein said compounds have at least two phenolic hydroxyl and/or amino groups, at least one aliphatic amino group, or at least one phenolic hydroxyl and/or amino group and at least one aliphatic amino group, and these groups are substituted with polyethylene glycol chains, whose degree of polymerization n is 5 to 250 and whose terminal hydroxyl group is substituted by C1-C12 alkyl ester or ether, each substance being substituted by at least two such polyethylene glycol chains. U.S. Pat. No. 5,622,685 also describes compounds containing PEG chains attached via linker wherein the polyethylene glycol chains are attached via biologically non-hydrolyzable or poorly hydrolyzable linkers.
The closest analogue to the present invention is the method disclosed in U.S. Pat. No. 6,147,207 by Sinn et al. entitled “Method for producing chlorins and bacteriochlorins containing polyether”. The method includes the bonding of a polyether to a porphyrin and conversion of the porphyrin containing polyether by means of a reducing agent. In the preferred embodiment of the invention the polyether is a polyethylene glycol (PEG). The key disadvantage of the method is that only a very limited number of compounds can be obtained due to the chemistry of the process. Also, the application (as well as other applications by Sinn et al.) discloses bis-, tris or tetra-PEGylated compounds, but not mono-PEGylated compounds. In practice it is possible to get only a complex mixture of isomers and oligomers of PEG-residue containing compounds. This fact makes reliable quantitative analysis and quality control, which are mandatory for the preparation of pharmaceutical products, practically impossible. Moreover, U.S. Pat. No. 6,147,207 discloses the preparation of products which bear PEG-chains which could be terminated only with OH and OMe groups, thus limiting the practical use of the obtained compounds.
Thus there is a need to provide new water-soluble mono-PEGylated tetrapyrrole derivatives for photodynamic therapy and to provide an easy and efficient method to produce such water-soluble mono-PEGylated compounds. The present invention addresses these needs.